2.1 Technical Field
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods. In particular, the invention relates to novel insulin-like growth factor-binding protein (IGFBP)-like polypeptides.
2.2 Background Art
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences. Proteins are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity. It is to these polypeptides and the polynucleotides encoding them that the present invention is directed. In particular, this invention is directed to novel IGFBP-like polypeptides and polynucleotides.
The IGFBP family of proteins has several members, including IGFBP-1 through IGFBP-7, which bind with high affinity to the insulin-like growth factors (IGFs), IGF-1 and IGF-II. Binding of IGFs by IGFBPs can modulate IGF activity, increase IGF serum half-life, and transport IGFs to appropriate sites. IGFBP-7(also known as mac25 and angiomodulin) demonstrates specific binding to IGFs, but with low affinity, and unlike the other members of this superfamily, is a high-affinity insulin binding protein, making it a strong therapeutic candidate in treating type I and type II diabetes mellitus. IGFBP-7 likely binds to insulin, thus increasing it stability and half life in the blood. (Yamanaka et al., J. Biol. Chem., (1997) 272(49):30729–30734). The IGFBPs differ by molecular weight, amino acid composition, distribution in biological fluids, and influence upon IGF activity. They share a highly conserved N-terminal and C-terminal domain that contain 12 and 6 cysteine residues, respectively, and a variable middle domain.
Approximately >97% of IGFs are bound by IGFBPs. IGFs are about 7.5-kDa single-chain protein homologues of insulin that can act locally, as autocrine or paracrine factors, or as endocrine growth factors that circulate in the plasma to act at distant sites. The IGFs can induce many responses that include mitogenesis within local tissue environments, induction of cellular differentiation, and metabolic effects such as increased amino acid uptake, and protein synthesis. They are synthesized and secreted by many tissues, although the primary sites of expression are liver, and to a lesser extent, bone.
IGFBP-3 is the major carrier protein for IGFs in the plasma and also modulates IGF receptor binding. IGFBP-3 binds to IGF in a 150-kDa trimeric complex that also contains the acid-labile subunit (ALS). These complexes function to extend the half-life of IGF in the plasma; the half-life of free IGF is 8–30 minutes, while that of IGF in the trimeric complex is about 15 hours. These complexes also provide a reservoir of IGF for target tissues. Limited proteolysis of IGFBP-3 results in a 50-fold lower affinity for IGF-I, allowing its release.
IGFBP-4 and -5 control IGF influence on bone and cartilage growth. Binding of IGF to IGFBP-5 reduces IGF growth-mediated activity. However, it also has the unique property of adhering to fibroblast extracellular matrix (ECM). When bound to ECM, the affinity of IGFBP-5 for IGF is decreased approximately 7-fold, suggesting that IGFBP-5 may deliver IGF to particular cell types, such as osteoblasts and cartilage, and potentiate its action at those sites.
IGFBP-6 also reduces IGF-I activity, and is associated with decreased steroidogenesis.
IGFBP-7 blocks insulin binding to the insulin receptor, and thereby inhibits the earliest steps in insulin action, such as autophosphorylation of the insulin beta subunit and phosphorylation of IRS-1. (Yoshitaka et al. Inhibition of Insulin Receptor Activiation by Insulin-like Growth Factor Binding Protein, J. Biol. Chem., (1997) 272(49): 30729–30734) Due to its ability to bind insulin with high affinity, IGFBP-7 might also be involved in pregnancy induced insulin resistance and type II diabetes mellitus.
Because the IGFs play a role in stimulating growth, their attenuation by IGFBP binding has been suggested as a mechanism to prevent tumor growth. For instance, increased concentrations of IGFBP-3 inhibit the proliferation of the breast cancer cell line, MCF7, and thus IGFBPs possibly work as antimitogens. Free IGFBP-3 may also bind to IGFBP-3 receptors on cancer cells and inhibit tumor cell growth, as well as induce apoptosis in an IGF-independent manner (Grimberg, A and Cohen P. J. Cell Physiol. (2000) 183:1–9). Circulating IGFBP-3 levels are also correlated with cancer risk. Prospective studies have shown that low levels of IGFBP-3 were associated with a doubled risk of prostate cancer, a fourfold increased risk of colorectal neoplasia, and a higher risk of breast and lung cancer (Giovannucci E. Horm Res. (1999) 3:34–41). Additionally, IGFBP-7 has been shown to be down-regulated at the transcription level in carcinoma cell lines, suggesting this member has a tumor suppressor activity. (Swisshelm et al. Proc. Natl. Acad. (1995) 92:4472–4476)
IGFs and IGFBPs are also involved in tissue remodeling. Because IGFBP-5 associates with the ECM and releases bound IGF at those sites, it induces tissue-specific cell proliferation and differentiation. In arthritis, proinflammatory cytokines such as TNF-α, IL-1α, and IL-1β cause the release of IGFBP-3, and IGFBP-5. These do not associate with the ECM and suppress IGF-I induced proteoglycan synthesis. Decreased proteoglycan synthesis coupled with degradation of cartilage matrix, allows breakdown of cartilage between joints. In addition, IGFBP-5 has also been implicated in bone remodeling, and tissue remodeling of the involuting mammary gland.
IGFBP-1 and IGFBP-3 also regulate wound healing. Nonphosphorylated IGFBP-1 enhances wound-breaking strength and re-epithelialization, a response that IGF alone cannot elicit. This suggests that IGFBP-1 accelerates wound healing by enhancing IGF-1 action, and may stimulate cell migration in an IGF-independent manner. Also, IGFBP-3 protease activity is increased following surgery and during chronic illnesses. Therefore, reductions in IGFBP-3 may allow increased IGF at tissue sites and contribute to increased metabolism and cellular division after insult.
IGFBPs also modulate the actions of IGFs on female reproductive function by synergizing with pituitary gonadotropins and ovarian steroid hormones. At various sites in the female reproductive tract, small changes (overproduction or deficiency) of IGFBPs may result in pathological conditions such as anovulation and hyperandrogenism, and inadequate differentiation of the endometrium (Wang, H.-S. and Chard, T. (1999) 161:1–13). During pregnancy, IGFBP-1 is an important modulator of IGF-1 activity. Maternal IGF-I promotes fetal growth and stimulates nutrient transport in the placenta. The presence of nonphosphorylated IGFBP-1, which has a decreased affinity for IGF-I, appears to enhance these activities of IGF-I and promote fetal growth. The IGFBPs are important modulators of IGFs. Without appropriate regulation of these factors, improper growth and differentiation of cells will result, possibly causing disease states.